Abstract

This unit presents a generalized protocol and describes the principles involved in cloning yeast genes by complementation in yeast. The protocol is presented using a hypothetical mutation of yeast, the cdc101-1 mutation. This mutation was isolated as a cell cycle mutant and is both recessive and temperature-sensitive for growth: it can grow relatively normally at 30 degrees C but is unable to make a colony at 37 degrees C. A genomic DNA clone that complements this mutation will be isolated by transforming the cdc101-1 strain with a yeast genomic library and subsequently screening for temperature-resistant colonies. Once isolated, two steps are necessary to prove that the insert present on the plasmid contains the wild-type CDC101 gene. First, segregation of the complementing plasmid must result in co-loss of both the plasmid-borne selectable marker and the complementing phenotype, demonstrating that the observed complementation is plasmid-specific and is not due to reversion of the cdc101-1 mutation. Second, it must be ruled out whether the cloned gene encodes a phenotypic suppressor of the mutation, rather than the wild-type gene. This is done via a complementation test, which demonstrates whether or not a disruption of the cloned gene that is integrated into the genome can complement the original mutation.